The yeasts have received broad attention in recent years, and offer some advantages of the prokaryotic system, such as simple genetic manipulation and rapid growth. The yeast organisms are able to make post-translational protein modifications typical of eukaryotic cells. Among those employed as expression systems of interest is Pichia pastoris, which has received wide acceptance for the production of biopharmaceuticals, since it is capable of doing some post-translational process, such as glycosylation, so very similar to mammals.
One of the most important features of Pichia pastoris is its ability to grow in culture medium containing methanol as the sole source of carbon. The first step in the use of methanol is the oxidation of this, leading to the formation of formaldehyde and hydrogen peroxide, a reaction catalyzed by the enzyme alcohol oxidase. Pichia pastoris has two genes that encode for alcohol oxidase, AOX1 and AOX2, but only the former is heavily regulated by methanol while the second is expressed in low quantities. When Pichia pastoris grows in the presence of glycerol, glucose or ethanol, the alcohol oxidase enzyme is not found, however, in the presence of methanol, the alcohol oxidase enzyme 1 (AOX1) may reach 35% of total cellular proteins. The control of AOX1 gene expression occurs at transcriptional level. The promoter of the gene AOX1 has been widely used for the construction of expression vectors of Pichia pastoris for being strong and highly adjustable, which reduces the possibility of toxic proteins harming the cell growth. These vectors are integrative type, allowing the stabilization of the exogenous gene message in the genome of Pichia pastoris. 
Another major advantage of expressing heterologous proteins or polypeptides as secreted recombinant proteins in Pichia pastoris is that Pichia pastoris secretes very low levels of native proteins. More interesting, there have been no reports of extracellular proteases in Pichia pastoris and studies suggest that most of the proteases active in Pichia pastoris are intracellular and not secreted (Jayanta Sinha et al., Biotechnology and Bioengineering, 89:102-112, 2005).
Still another important feature is that the expression of heterologous proteins or polypeptides in Pichia pastoris has shown high levels of production when grown at high cell densities in simple defined medium. Indeed, the strong promoter, coupled with the high cell density fermentation, has allowed production of heterologous proteins or polypeptides at high concentration.
Furthermore, it is known from U.S. Pat. No. 5,691,166 (Martin A. Gleeson et al.) that while growth at high cell density enables the production of heterologous proteins or polypeptides in remarkably high yields, growth at high cell density also provides for a relatively high level of vacuolar proteases in the fermentation media (since about 1% of cells typically undergo lysis during yeast fermentation, the high cell density process is accompanied by the release of substantial quantities of cellular material into the media, including vacuolar proteases). Therefore, during the production of heterologous proteins or polypeptides in a high cell density process, some of the secreted heterologous proteins or polypeptides produced by Pichia could be subjected to substantial proteolysis.
Indeed it is well known that generally in yeast, the major store of proteolytic activity is located within the lumen of the vacuolar compartment (Jones, Methods Enzymol 194:428-453, 1991). These proteases are released into the fermentation broth by spontaneous and inevitable cell lysis. Although vacuolar proteases are required for several developmental transitions in the life cycle of yeast cells (e.g., sporulation), they are dispersible for vegetative growth. The majority of vacuolar proteases are synthesized and transported through the secretory pathway as enzymatically inactive zymogens (Klionsky et al., Microbiol. Rev. 54:266-292, 1990; Raymond et al., Int. Rev. Cytol. 139:59-120, 1992). They are proteolytically activated by the combined action of proteinase A, the product of the PEP4 gene, and proteinase B, the product of the PRB1 gene.
Many other documents of the prior art also disclose that the proteolytic degradation of secreted heterologous proteins or polypeptides in Pichia pastoris is indeed due to the release of proteases in the culture medium caused by degradation of the cell membrane due to cell lysis in high cell density fermentation (Zhengjun Li et al., Protein Expression and Purification, 21:438-445, 2001; Jayanta Sinha et al., Biotechnology and Bioengineering, 89:102-112, 2005; Diethard Mattanovich et al., Microbial Cell Factories, 8:29, 2009).
In view of the excellent performance of the Pichia-based expression system for the production of many heterologous proteins or polypeptides, it would be desirable to reduce or eliminate the proteolytic activities of Pichia pastoris. This would reduce the likelihood of degradation of protease-sensitive proteins when produced in recombinant Pichia hosts, allow an enhanced ability to express and recover heterologous proteins or polypeptides in substantially intact form and avoid endogenous protease contaminants, further allowing easier purification of heterologous proteins, such as heterologous proteases.
Various techniques attempt to deal with this problem of the proteolytic degradation of secreted heterologous proteins or polypeptides in Pichia pastoris. For example, one could modify the conditions under which recombinant Pichia strains are grown so as to inhibit protease activity. This could be accomplished, for example, by adjusting the pH of the medium sufficiently to inhibit the action of various proteases. This approach, however, may affect the ability of the host organism to express certain heterologous proteins or polypeptides (as well as the stability of the resulting protein, once expressed). Alternatively it is possible to add protease inhibitors.
Another known example is the use of protease-deficient strains. Some vacuolar protease-deficient strains of Pichia are already available, such as Pichia methanolica strain disclosed in WO 99/14347 (Zymogenetics, Inc.), which is deficient in proteinase A or proteinase B activity, or Pichia pastoris strains SMD1168, SMD1163 or SMD1165, which posses a substantial decrease in or elimination of proteinase A, carboxypeptidase Y and/or proteinase B activities.
However, it is not easy to create protease-deficient strains. Indeed the proteolytic processes in eukaryotic organisms are quite complicated and involved in cell metabolism. Thus, it is not possible to predict if elimination and/or modification of one or more of the enzyme(s) that are involved in proteolytic processing pathways will have an impact on the viability of the host cells, and/or the stability of the secreted heterologous protein or polypeptide.
The drawback to each of the above-mentioned attempts is that the degradation of secreted heterologous proteins or polypeptides in Pichia pastoris is still observed.
The problem of the degradation of secreted heterologous proteins or polypeptides in Pichia pastoris remains unsolved. Surprisingly the Applicant was able to overcome this problem in the present invention.